The peroxisome is a ubiquitous organelle of eukaryotic cells. It participates in a wide variety of metabolic processes and defects in peroxisome biogenesis are the cause of numerous human diseases. The peroxisome biogenesis disorders are one of these. These lethal inherited disorders are caused by defects in the biogenesis of the peroxisomes, which in turn results in the loss of virtually all peroxisomal metabolic functions. We and others have recently found that the proteins PEX3, PEX16 and PEX19 are required for the biogenesis of peroxisome membranes and that PEX11 proteins participate in the division of peroxisomes. These and other results have led to the proposal of a two pathway model of peroxisome biogenesis and the long-term objective of this proposal is to test this model of peroxisome membrane biogenesis. This will be accomplished through four specific aims. The first will involve the analysis of peroxisome membrane synthesis as mediated by PEX3, PEX16, and PEX19. The second aims involves an analysis of how PEX11a and PEX11b mediate peroxisome division. The third will involve the development of triply mutated cell lines for use in aim 1. The fourth aim will be to identify the targeting signals that direct integral PMPs to the peroxisome and into the peroxisome membrane. The significance of these studies is that they will improve our understanding of eukaryotic cell biology and the molecular basis of human disease by testing our current hypotheses about the mechanisms of peroxisome membrane biogenesis.